FIGURE 6.

Selective up-regulation of HIV-1 RNA levels by an intact UPF1 protein. Cells were (lane 1) mock transfected or co-transfected with (lane 2) HIV-1 proviral DNA (pNL4-3, HIV-1) and UPF1^WT, (lane 3) an expression construct for UPF3b with a λN-N-terminal tag (λN/UPF3b), (lane 4) TDN (R844C), or Flag-tagged expression constructs coding for deletion mutants of UPF1 including (lane 5) the C-terminal deletion mutant, UPF1^I–1074, (lane 6) the internally deleted UPF1^Δ20–150, and (lane 7) the N-terminal deleted UPF1^ΔN40. At 30 h post-transfection, cells were harvested for Western blot analysis for UPF1, UPF3b, and GAPDH (loading control). RNA was also isolated from cell extracts, and Northern blotting was performed to measure steady-state levels of HIV-1 and gapdh mRNA. Because the primary anti-UPF1 antibody did not efficiently recognize the truncation and deletion mutants, Western blotting using an anti-Flag antibody was employed to identify the Flag-tagged UPF1 proteins expressed in trans (bottom panel). The percentages represent genomic RNA levels related to the signal obtained in UPF1^WT and HIV-1-expressing cells. The results shown are representative of three independently performed experiments.